NALP7-based diagnosis of female reproductive conditions

ABSTRACT

Methods, reagents and kits are described for the diagnosis of a female reproductive condition, based on the detection of an alteration in a NALP7-encoding nucleic acid or a NALP7 polypeptide, relative to a corresponding wild-type NALP7-encoding nucleic acid or NALP7 polypeptide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Phase Application of PCT InternationalApplication No. PCT/CA2006/001256, filed Aug. 3, 2006, which waspublished in English under PCT Article 21(2) as InternationalPublication No. WO 2007/014463. This application further claims thebenefit of U.S. Provisional Patent Application No. 60/704,896 filed Aug.3, 2005. All of these applications are incorporated herein by referencein their entirety.

REFERENCE TO SEQUENCE LISTING

Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewithas an ASCII compliant text file named “Sequence Listing.txt” which wascreated on Feb. 22, 2011 and has a size of 77,390 bytes. The content ofthe aforementioned file named “Sequence listing.txt” is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods and reagents for the diagnosisof conditions of the female reproductive system.

BACKGROUND OF THE INVENTION

A number of female reproductive conditions exist which not only haveadverse effects on fertility, but may pose serious health concerns tofemale sufferers, such as cancer. Such conditions include gestationaltrophoblastic diseases, such as the phenomenon of recurrent hydatidiformmoles (HM), an abnormal human pregnancy with no embryo and cysticdegeneration of placental villi. Recurrent HM is a rare clinical entityin which molar tissues are diploids and have a biparental contributionto their genome. In a number of cases this condition has been observedto have a familial basis. Recurrent hydatidiform molar tissues areundistinguishable at both gross morphology and histopathology levelsfrom the common non-recurrent moles, which are androgenetic in most ofthe cases (80% of the cases), but may also be biparental (in 20% of thecases). The common form of hydatidiform moles occur in 1 in every 1500pregnancies in western countries, but at a higher incidence in the FarEast, Africa and Central America where the incidence of this conditionmay reach 1 in 100 pregnancies. Epidemiological studies performed tocorrelate this higher incidence with various environmental factorsfailed to reach significant conclusions, but shows a higher risk ofhydatidiform moles at the beginning and end of a woman's reproductivecycle. In addition, the relative risk of developing a second HM after aprevious molar pregnancy is 20 to 40 times the incidence of moles in thegeneral population indicating genetic susceptibility to moles.

In mammals, maternal effect genes, in addition to those coding foroocyte mRNAs and proteins that accumulate in the egg during oogenesis,extend to genes required in the maternal reproductive tract for normalpreimplantation and implantation development. Applicant has previouslymapped a genetic region responsible for recurrent HMs to a 15-cMinterval on 19q13.4 in two unrelated families, MoLb1 and MoGe2 (Moglabeyet al., 1999). Additional families from various ethnic groups werereported and most of them were found linked to 19q13.4, indicating amajor locus in this region leading to recurrent HMs. The analysis ofthese families narrowed down the HM candidate region to a 1.1-Mbinterval (Sensi et al. 2000; Hodges et al. 2003).

Therefore, there is a continued need to identify the gene associatedwith such disorders.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention relates to NALP7 and conditions of the female reproductivesystem, including diagnosis of such conditions based on NALP7.

Accordingly, In a first aspect, the invention provides a method fordiagnosing a reproductive condition or a predisposition for areproductive condition in a female (e.g., human) subject, the methodcomprising detecting an alteration in the sequence of the NALP7 gene orthe sequence of its mRNA or encoded polypeptide in a tissue sample fromsaid subject relative to the sequence of the wild-type NALP7 gene or thesequence of its mRNA or encoded polypeptide, wherein said alterationindicates that the subject suffers from or has a predisposition for thereproductive condition. In embodiments, the reproductive condition isselected from gestational trophoplastic disease, gestationaltrophoblastic tumor, hydatidiform mole, molar pregnancy, biparentalmolar pregnancy, androgenetic molar pregnancy, invasive mole,choriocarcinoma, premature ovarian failure, infertility, endometriosis,implantation failure, blighted ovum, recurrent spontaneous abortions,and preeclampsia.

In embodiments, the alteration is associated with altered splicing of aNALP7 transcript, such as altered splicing of exon 3, exon 7, or both,of said NALP7 gene.

In an embodiment, the method further comprises amplification of anucleic acid sequence suspected of comprising the alteration in thesample prior to the detection of the alteration.

In embodiments, detection of the alteration is performed using a methodselected from: (a) sequencing of the NALP7 nucleic acid sequence; (b)hybridization of a nucleic acid probe capable of specificallyhybridizing to a NALP7 nucleic acid sequence comprising the alterationand not to a corresponding wild-type NALP7 nucleic acid sequence; (c)restriction fragment length polymorphism analysis (RFLP); (d) amplifiedfragment length polymorphism PCR (AFLP-PCR); (e) amplification of anucleic acid fragment comprising a NALP7 nucleic acid sequence using aprimer specific for the alteration, wherein the primer produces anamplified product if the alteration is present and does not produce thesame amplified product when a corresponding wild-type NALP7 nucleic acidsequence is used as a template for amplification; (f) sequencing of theNALP7 polypeptide; (g) digestion of the NALP7 polypeptide followed bymass spectrometry or HPLC analysis of the peptide fragments, wherein thealteration of the NALP7 polypeptide results in an altered massspectrometry or HPLC spectrum as compared to wild-type NALP7polypeptide; and (h) immunodetection using an immunological reagentwhich exhibits altered immunoreactivity with a NALP7 polypeptidecomprising the alteration relative to a corresponding wild-type NALP7polypeptide.

In an embodiment, the method further comprises determining cytokinerelease of an immune cell of said subject, wherein a decrease incytokine release relative to a control level of cytokine release isfurther indicative that the subject suffers from or has a predispositionfor the reproductive condition.

In embodiments, the control level of cytokine release is selected froman established standard and a level of cytokine release of an immunecell comprising a wild-type NALP7 nucleic acid.

In an embodiment, the method further comprises selecting a prophylacticor therapeutic course of action in accordance with the detectedalteration.

In a further aspect, the invention provides a nucleic acid probe capableof specifically hybridizing to an altered NALP7 nucleotide sequence andnot to a corresponding wild-type NALP7 nucleotide sequence.

The invention further provides a primer or an amplification pair capableof specifically producing an amplified product from a templatecomprising an altered NALP7 nucleotide sequence and which does notproduce the same amplified product from a template comprising acorresponding wild-type NALP7 nucleotide sequence. In embodiments, theprimer or amplification pair are selected from SEQ ID NOs: 6-42.

The invention further provides an isolated altered NALP7 nucleic acid orfragment thereof, wherein said altered NALP7 nucleic acid or fragmentthereof comprises a nucleotide sequence comprising an alterationrelative to the nucleotide sequence of a wild-type NALP7 nucleic acid orfragment thereof.

The invention further provides an isolated nucleic acid comprising asequence that encodes an altered NALP7 polypeptide or fragment thereof.

The invention further provides an isolated nucleic acid comprising analteration described herein and which is substantially identical to orsubstantially complementary to the above-mentioned isolated nucleicacid.

In an embodiment, the nucleic acid comprises an altered NALP7 nucleotidesequence comprising an alteration associated with altered splicing of aNALP7 transcript, such as altered splicing of exon 3, exon 7, or both,of said NALP7 gene.

In an embodiment, the alteration occurs at a splice donor site, such asat the splice donor site at the boundary of exon 3 and intron 3, thesplice donor site at the boundary of exon 7 and intron 7, or both, ofthe NALP7 gene.

In an embodiment, the alteration results in a loss of a cleavage sitefor a restriction endonuclease (e.g., BstN1) in the NALP7 gene.

In an embodiment, the alteration is at an amino acid position within theNALP7 polypeptide selected from position 693, 399, 379, 99 and 657 ofthe NALP7 polypeptide.

In embodiments, the alteration is selected from a substitution of the Ccorresponding to the first position of the codon for Arg 693 of theNALP7 polypeptide and a substitution of the G corresponding to thesecond position of the codon for Arg 693 of the NALP7 polypeptide. Infurther embodiments, the alteration is selected from a substitution ofArg 693 with Trp (R693W).

In further embodiments, the alteration is selected from (a) asubstitution of Cys 399 with Tyr (C399Y); (b) a substitution of Lys 379with Asn (K379N); (c) a substitution of the codon for Glu 99 with a stopcodon (E99X); and (d) a substitution of Asp 657 with Val (D657V).

In embodiments, the alteration is selected from: (a) a substitution of Gwith A at the splice donor site at the boundary of exon 3 and intron 3(IVS3+1G>A); (b) a substitution of G with A at the splice donor site atthe boundary of exon 7 and intron 7 (IVS7+1G>A); (c) a substitution of Cwith T corresponding to the first position of the codon for Arg 693 ofthe NALP7 polypeptide; (d) a substitution of G with A corresponding tothe second position of the codon for Cys 84 of the NALP7 polypeptide;(e) a substitution of G with A corresponding to the second position ofthe codon for Cys 399 of the NALP7 polypeptide; (f) a substitution of Gwith C corresponding to the third position of the codon for Lys 379 ofthe NALP7 polypeptide; (g) a substitution of G with T corresponding tothe first position of the codon for Glu 99 of the NALP7 polypeptide; and(h) a substitution of A with T corresponding to the second position ofAsp 657 of the NALP7 polypeptide

The invention further provides a replicative cloning vector comprisingthe above-mentioned nucleic acid and a replicon operative in a hostcell.

The invention further provides a vector (e.g., an expression vector)comprising the above-mentioned nucleic acid operably linked to atranscriptionally regulatory element.

The invention further provides a host cell transformed with theabove-mentioned vector, replicative cloning vector or expression vector.

The invention further provides an isolated, recombinant or substantiallypure altered NALP7 polypeptide encoded by the above-mentioned nucleicacid.

The invention further provides a polypeptide comprising an alterationdescribed herein and which is substantially identical to theabove-mentioned isolated, recombinant or substantially pure alteredNALP7 polypeptide.

The invention further provides an antibody that binds specifically bindsthe above-mentioned altered NALP7 polypeptide.

The invention further provides an antibody capable of alteredimmunoreactivity with a NALP7 polypeptide comprising the alterationrelative to a corresponding wild-type NALP7 polypeptide, such as anantibody that selectively binds to the altered NALP7 polypeptide butdoes not bind to or binds to a lesser extent to a correspondingwild-type NALP7 polypeptide under the same conditions.

The invention further provides a kit for diagnosing a reproductivecondition or a predisposition for a reproductive condition in a femalesubject, said kit comprising means for detection of an alteration in thesequence of a NALP7 gene or the sequence of its mRNA or encodedpolypeptide in a tissue sample from said subject relative to thesequence of a corresponding wild-type NALP7 gene or the sequence of itsmRNA or encoded polypeptide. In embodiments, such means are chosen fromreagents for: (a) sequencing of the NALP7 nucleic acid sequence; (b)hybridization of a nucleic acid probe capable of specificallyhybridizing to a NALP7 nucleic acid sequence comprising the alterationand not to a corresponding wild-type NALP7 nucleic acid sequence; (c)restriction fragment length polymorphism analysis (RFLP); (d) amplifiedfragment length polymorphism PCR (AFLP-PCR); (e) amplification of anucleic acid fragment comprising a NALP7 nucleic acid sequence using aprimer specific for the alteration, wherein the primer produces anamplified product if the alteration is present and does not produce thesame amplified product when a corresponding wild-type NALP7 nucleic acidsequence is used as a template for amplification; (f) sequencing of theNALP7 polypeptide; (g) digestion of the NALP7 polypeptide followed bymass spectrometry or HPLC analysis of the peptide fragments, wherein thealteration of the NALP7 polypeptide results in an altered massspectrometry or HPLC spectrum as compared to wild-type NALP7polypeptide; and (h) immunodetection using an immunological reagentwhich exhibits altered immunoreactivity with a NALP7 polypeptidecomprising the alteration relative to a corresponding wild-type NALP7polypeptide. In embodiments, the reagents are chosen from theabove-mentioned antibody, primer (or pair), and probe.

In an embodiment, the kit further comprises means to determine cytokinerelease of an immune cell of said subject.

In an embodiment, the kit further comprises instructions for diagnosinga reproductive condition or a predisposition for a reproductivecondition in a female subject.

The Invention further provides a method of identifying a compound forrestoring defective immune function associated with a reproductivecondition, said method comprising determining whether cytokine releaseof an immune cell comprising an altered NALP7 nucleic acid orpolypeptide is increased in the presence of a test compound relative toin the absence of said test compound; wherein said increase isindicative that said test compound may be used for restoring defectiveimmune function associated with a reproductive condition.

In an embodiment, the immune cell is a a peripheral blood mononuclearcell (PBMC). Iin a further embodiment, the immune cell is a lymphocyteor monocyte.

In embodiments, the cytokine is selected from interleukin-1β (IL-1β) andTNF alpha (TNFα).

Other advantages and features of the present invention will become moreapparent upon reading of the following non-restrictive description ofspecific embodiments thereof, given by way of example only withreference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Partial pedigree of family MoLb1 showing the limit of theproximal boundary of the hydatidiform mole candidate locus on 19q13.4.Markers are ordered (top to bottom) from centromere to telomere andtheir positions are given in contig NT_(—)011109.15. Genotyping wasperformed using publicly available and newly generated microsatellitemarkers by incorporation of radiolabelled nucleotides in the PCRamplification and separation of the products on 5% denaturingpolyacrylamide gels. Black symbols indicate affected women, whitesymbols unaffected, and shaded symbol indicates a woman with unknowndisease status. The homozygous region in the three affected sisters isindicated. The black box shows the region that is homozygous in eachpatient. The proximal border of the candidate region is defined bymarker 11515_(—)31 due to its heterozygosity in patient 4.

FIG. 2: Pedigree of family MoPa61 with recurrent hydatidiform moles.Twenty-three informative microsatellite markers were genotyped todetermine linkage to 19q13.4. Markers are ordered (top to bottom) fromcentromere to telomere and are indicated on the left along with theirposition in contig NT_(—)011109.15. These data define marker COX6B2 asthe distal boundary of the HM candidate region due to its lack ofhomozygosity in all three affected sisters.

FIG. 3: Segregation of the IVS3+1G>A mutation in family MoLb1. a,sequence electropherogram showing the exon3/intron3 boundary in normalcontrol and in patient MoLb1-4. The recognition site of the restrictionenzyme BstN1, CCWGG, is abolished by the splice mutation IVS3+1G>A.Normal control sequence: AGCCAGGTGGGTA (SEQ ID NO: 43); MoLb1-4 patientsequence: AGCCAGATGGGTA (SEQ ID NO: 44). b, Partial pedigree of MoLb1showing the genotypes of the different members for the IVS3+1 mutation.The band at 206 bp is uncut by BstN1 and thus contains the mutation, theband at 153 bp resulted from the digestion of the normal allele withBstN1. The parents (1, 2, and 3) are heterozygous for the mutation, theaffected women, 4, 6, and 8, are homozygous for the mutation, theunaffected sister, 7, is homozygous for the normal allele, as is 5 whosestatus with respect to molar pregnancy is unknown. Member 9 is a carrierfor the mutation and her phenotype is also unknown.

FIG. 4: DNA sequence electrophorograms showing the IVS7+1 G>A, R693Wmutations. For each mutation, the control individuals homozygous for thenormal alleles are shown at the top; the mothers of the patients who areheterozygous for the normal and mutant alleles are shown in the middle;and affected females, homozygous for the mutations are shown at thebottom. The pedigree symbols are as described in the legend of FIG. 1.

FIG. 5: Abnormal RNA splicing resulting from IVS7+1G>A mutations on RNAextracted from EBV-transformed lymphoblastoid cell lines from thepatients. RT PCR using primers located in exons 6 and 8 of NALP7 in onepatient from family MoPa61 amplified a ˜1 kb fragment present only inthe patient from MoPa61, but not in a patient from MoLb1 (with IVS+1G>A)or in control. The ZNF28 gene was amplified on the same samples to showthe equal amount of cDNA.

FIG. 6: Genomic DNA sequence of human NALP7 (SEQ ID NO: 1; derived fromGenBank accession No. NT_(—)011109.15)

FIG. 7: DNA (SEQ ID NO: 2) and polypeptide (SEQ ID NO: 3) sequence ofhuman NALP7, 980 amino acid isoform (GenBank accession No. AY154462 orNM_(—)206828). Coding sequence is defined by position 71-3013 of DNAsequence.

FIG. 8: DNA (SEQ ID NO: 4) and polypeptide (SEQ ID NO: 5) sequence ofhuman NALP7, 1009 amino acid isoform (GenBank accession No.NM_(—)139176). Coding sequence is defined by position 71-3100 of DNAsequence.

FIG. 9: IL-1β secretion by PBMCs with NALP7 mutations. Blood wascollected from one patient from family MoLb1 (Lb1-4 in the right panel)and three patients from MoGe2 (Il-2, Il-3, Il-8, left panel). PBMCs wereisolated from blood using the Ficoll gradient technique, 500,000cells/well were stimulated with 100 ng/mL of LPS, supernatant wascollected 20 hours later and IL-1β levels at the indicated dilutionswere measured using ELISA.

FIG. 10: TNFα secretion by PBMCs with NALP7 mutations. TNFα levels weremeasured as described in Example 8 below.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In the studies described herein, applicant has identified a defectivematernal gene, NALP7, and its causative role in different conditionsaffecting the female reproductive system, such as recurrent molarpregnancies.

NALP7 is one of 14 members of the NALP proteins, a large subfamily ofthe CATERPILLER protein family involved in inflammation and apoptosis.NALP7 is related to the mouse MATER, (also a member of the CATERPILLERprotein family). The NALP7 gene consists of 11 exons encoding for 1009amino acid protein (the longest isoform). Three transcriptional isoformsNALP7V1-V3 involving the alternative splicing of exons 5, 9, and 10 havebeen described (Okada et al., 2004). NALP7 contains an amino-terminalPYRIN domain (PYD) (also called DAPIN), a putative protein-proteininteraction domain found in all the CATERPILLER protein family andthought to function in apoptotic and inflammatory signaling pathways; aNACHT domain found in neuronal apoptosis inhibitor proteins as well asin those involved in the major histocompatibility complex (MHC) class IItransactivation and caspase-recruitment proteins; a nuclear localizationsignal (NLS) present within the NACHT domain; and 9 to 10 leucine-richrepeats (LRRs) (depending on the splicing isoforms) found in the RanGTPase activating proteins (RanGAP1), highly conserved proteinsessential for nuclear transport, cell cycle regulation, mitotic spindleformation, and post mitotic nuclear envelope assembly. NALP7 has beenshown to inhibit caspase-1 dependent IL-1β secretion, which in turninduces NALP7 expression. NALP7 (also referred to as PYPAF3) wasrecently shown to be upregulated in testicular seminoma tumors where itsdown regulation by transfection with small interfering RNA results ingrowth suppression (Okada et al., 2004; International patent applicationpublication no. WO2004/031410 [Nakamura et al., Apr. 15, 2004]).

As described herein, applicants have identified a number of mutations inthe NALP7 gene in families having female members suffering fromreproductive conditions, such as recurrent hydatidiform moles. Suchidentified mutations include:

-   a) a substitution of G with A in the GT sequence of the splice donor    site at the boundary of exon 3 and intron 3 (IVS3+1G>A) of the NALP7    gene;-   b) a substitution of G with A in the GT sequence of the splice donor    site at the boundary of exon 7 and intron 7 (IVS7+1G>A) of the NALP7    gene;-   c) a substitution of C with T corresponding to the first position of    the codon for Arg 693 of the NALP7 polypeptide;-   d) a substitution of G with A corresponding to the second position    of the codon for Cys 84 of the NALP7 polypeptide,-   e) a substitution of G with A corresponding to the second position    of the codon for Cys 399 of the NALP7 polypeptide,-   f) a substitution of G with C corresponding to the third position of    the codon for Lys 379 of the NALP7 polypeptide,-   g) a substitution of G with T corresponding to the first position of    the codon for Glu 99 of the NALP7 polypeptide; and-   (h) a substitution of A with T corresponding to the second position    of Asp 657 of the NALP7 polypeptide.

The above mutations (a) and (b) have resulted in incorrect splicing ofthe NALP7 transcript, notably in respect of the exon 3/intron 3 boundaryand the exon 7/intron 7 boundary, respectively. For example of incorrectsplicing in case of (b), the mutation was shown to result in theinclusion of the entire intron 7 resulting in the addition of one aminoacid (a serine) to exon 7, followed by a stop codon, resulting thereforein a shortened protein of 824 amino acids.

The above mutation (a) has also resulted in a loss of a cleavage site ofthe restriction endonuclease BstN1.

The above mutation (c) has resulted in an alteration at Arg 693 of theNALP7 polypeptide sequence, notably its substitution with Trp. The abovemutation (d) has resulted in an alteration at Cys 84 of the NALP7polypeptide sequence, notably its substitution with Tyr. The abovemutation (e) has resulted in an alteration at Cys 399 of the NALP7polypeptide sequence, notably its substitution with Tyr. The abovemutation (f) has resulted in an alteration at Lys 379 of the NALP7polypeptide sequence, notably its substitution with Asn. The abovemutation (g) has resulted in an alteration at Glu 99 of the NALP7polypeptide sequence, notably its substitution with a stop codon. Theabove mutation (h) has resulted in an alteration at Asp 657 of the NALP7polypeptide, notably its substitution with a Val.

Applicant has further shown herein NALP7 transcription in EBVlymphoblastoid cell lines, normal human uterus, ovaries, unfertilizedoocytes at the germinal vesicle and metaphase I stages, early embryocleavage (1 to 6 cells) and first trimester chorionic villi at 6 and 12weeks of gestation.

Accordingly, in an aspect, the invention relates to NALP7-baseddiagnosis of conditions of the female reproductive system. The inventionthus provides methods and reagents to detect an alteration in NALP7 orits encoded polypeptide, including an alteration in its nucleic acidsequence (including its DNA, mRNA (or cDNA)) or polypeptide sequence, ina sample from a female subject. The presence of an alteration relativeto the corresponding wild-type nucleic acid sequence or polypeptidesequence is indicative that the female subject suffers from or has apredisposition for the reproductive condition. The invention furtherrelates to screening to identify compounds capable of restoringdefective immune function associated with a female reproductivecondition, e.g., that associated with mutant NALP7.

The invention thus provides a method for diagnosing a reproductivecondition or a predisposition for a reproductive condition in a femalesubject, the method comprising detecting an alteration in the sequenceof the NALP7 gene or the sequence of its mRNA or encoded polypeptide ina tissue sample from said subject relative to the sequence of thewild-type NALP7 gene or the sequence of its mRNA or encoded polypeptide.The presence of the alteration indicates that the subject suffers fromor has a predisposition for the reproductive condition.

The invention further provides an in vitro method for diagnosing areproductive condition or a predisposition for a reproductive conditionin a female subject, the method comprising detecting an alteration inthe sequence of the NALP7 gene or the sequence of its mRNA or encodedpolypeptide in a tissue sample from said subject relative to thesequence of the wild-type NALP7 gene or the sequence of its mRNA orencoded polypeptide. The presence of the alteration indicates that thesubject suffers from or has a predisposition for the reproductivecondition.

Examples of wild-type NALP7 DNA and polypeptide sequences are providedin FIGS. 6-8 and SEQ ID NOs 1, 2 and 4 (DNA) and SEQ ID NOs 3 and 5(polypeptide).

Applicant has further described herein a decrease in cytokine release inimmune cells obtained from a patient harboring a NALP7 mutation.Accordingly, in an embodiment, the above-mentioned method furthercomprises determining cytokine release of an immune cell of saidsubject, wherein a decrease in cytokine release relative to a controllevel of cytokine release is further indicative that the subject suffersfrom or has a predisposition for the reproductive condition.

The above-mentioned control level of cytokine release may be for examplean established standard (e.g., a level established in the art for animmune cell capable of wild-type, normal or healthy immune function) ora level of cytokine release of an immune cell comprising a wild-typeNALP7 nucleic acid or polypeptide.

The above-mentioned immune cell may be for example a peripheral bloodmononuclear cell (PBMC), lymphocyte, or monocyte.

In embodiments, the above-mentioned cytokine is selected frominterleukin-1β (IL-1β) and TNF alpha (TNFα).

In an embodiment, the subject is a female mammal, e.g., a human femalesubject.

In embodiments, the reproductive condition is selected gestationaltrophoplastic disease, gestational trophoblastic tumor, hydatidiformmole, molar pregnancy, biparental molar pregnancy, androgenetic molarpregnancy, invasive mole, choriocarcinoma, premature ovarian failure,infertility, endometriosis, implantation failure, blighted ovum,recurrent spontaneous abortions, preeclampsia, and stillbirth.

In various embodiments, the above noted tissue sample comprises a tissueor body fluid from the subject, such as blood, serum, lymphocytes,epithelia, endometrial and uterine biopsies, and oocytes.

“Alteration” as used herein in respect of a nucleotide or polypeptidesequence refers to any type of mutation or change relative to thecorresponding wild-type nucleotide or polypeptide sequence, includingdeletions, insertions, substitutions and point mutations. In the case ofa nucleotide sequence, such an alteration may occur in coding and/ornon-coding regions. Mutations of a nucleotide sequence may for exampleresult in the creation of a stop codon, frameshift mutation, alteredsplicing or an amino acid substitution. In the case of mutations in aregulatory region (e.g., a promoter), a decrease or loss of mRNAexpression may result. Accordingly, in various embodiments, thealteration is selected from a deletion from, substitution of and/orinsertion into a NALP7 nucleic acid and/or polypeptide sequence.

In an embodiment, the alteration results in altered splicing relative towild-type NALP7. Such altered splicing may occur in respect of exon 3and/or exon 7. In embodiments, the alteration may occur in the splicedonor site, such as in the GT splice donor sequence.

In an embodiment the alteration results in altered sensitivity to arestriction endonuclease, such as a loss of a cleavage site for arestriction endonuclease. In an embodiment, the restriction endonucleaseis BstN1.

In an embodiment, the alteration occurs at position 693 of the NALP7polypeptide, in further embodiments, the alteration is a substitution ofArg 693 with a different amino acid, such as Trp.

In an embodiment, the alteration occurs at position Cys 84 of the NALP7polypeptide, in further embodiments, the alteration is a substitution ofCys 84 with a different amino acid, such as Tyr.

In an embodiment, the alteration occurs at position 399 of the NALP7polypeptide, in further embodiments, the alteration is a substitution ofCys 399 with a different amino acid, such as Tyr.

In an embodiment, the alteration occurs at position 379 of the NALP7polypeptide, in further embodiments, the alteration is a substitution ofLys 379 with a different amino acid, such as Asn.

In an embodiment, the alteration occurs at position 99 of the NALP7polypeptide, in further embodiments, the alteration is a substitution ofGlu 99, such as with a stop codon

In an embodiment, the alteration occurs at position 657 of the NALP7polypeptide, in further embodiments, the alteration is a substitution ofAsp 657 with a different amino acid, such as Val.

In an embodiment, the alteration occurs at a splice donor and/or spliceacceptor site. In an embodiment, the alteration occurs at a splice donorsite at the boundary of exon 3 and intron 3, in a further embodiment, ata splice donor site at the boundary of exon 7 and intron 7.

In further embodiments, the alteration is selected from (a) asubstitution of G with A in the GT sequence of the splice donor site atthe boundary of exon 3 and intron 3 (IVS3+1G>A) of the NALP7 gene; (b) asubstitution of G with A in the GT sequence of the splice donor site atthe boundary of exon 7 and intron 7 (IVS7+1G>A) of the NALP7 gene; (c) asubstitution of C with T corresponding to the first position of thecodon for Arg 693 of the NALP7 polypeptide; (d) a substitution of G withA corresponding to the second position of the codon for Cys 84 of theNALP7 polypeptide; (f) a substitution of G with C corresponding to thethird position of the codon for Lys 379 of the NALP7 polypeptide; (g) asubstitution of G with T corresponding to the first position of thecodon for Glu 99 of the NALP7 polypeptide; and (h) a substitution of Awith T corresponding to the second position of Asp 657 of the NALP7polypeptide.

The above-noted alteration is relative to a wild-type NALP7 sequence,examples of which are provided in FIGS. 6-8 and SEQ ID NOs 1, 2 and 4(DNA) and SEQ ID NOs 3 and 5 (polypeptide). The invention furtherprovides an isolated nucleic acid or polypeptide comprising annucleotide or amino acid sequence selected from SEQ ID NOs 1, 2 and 4(DNA) and SEQ ID NOs 3 and 5 (polypeptide) further comprising analteration noted herein or any combination of the alterations notedherein.

The detection of any combination of the above-noted alterations may alsobe used in the methods of the invention.

Further, the above-mentioned method may further comprise selection of aprophylactic or therapeutic course of action in accordance with thedetected alteration.

The above noted alteration may be detected by a number of methods whichare known in the art. Examples of suitable methods include sequencing ofthe NALP7 nucleic acid sequence; hybridization of a nucleic acid probecapable of specifically hybridizing to a NALP7 nucleic acid sequencecomprising the alteration and not to (or to a lesser extent to) acorresponding wild-type NALP7 nucleic acid sequence (under comparablehybridization conditions); restriction fragment length polymorphismanalysis (RFLP); Amplified fragment length polymorphism PCR (AFLP-PCR);amplification of a nucleic acid fragment comprising a NALP7 nucleic acidsequence using a primer specific for the alteration, wherein the primerproduces an amplified product if the alteration is present and does notproduce the same amplified product when a corresponding wild-type NALP7nucleic acid sequence is used as a template for amplification (e.g.allele-specific PCR); sequencing of the NALP7 polypeptide; Digestion ofthe NALP7 polypeptide followed by mass spectrometry or HPLC analysis ofthe peptide fragments, wherein the alteration of the NALP7 polypeptideresults in an altered mass spectrometry or HPLC spectrum as compared towild-type NALP7 polypeptide; and immunodetection using an immunologicalreagent (e.g. an antibody, a ligand) which exhibits alteredimmunoreactivity with a NALP7 polypeptide comprising the alterationrelative to a corresponding wild-type NALP7 polypeptide; Immunodetectioncan measure the amount of binding between a polypeptide molecule and ananti-protein antibody by the use of enzymatic, chromodynamic,radioactive, magnetic, or luminescent labels which are attached toeither the anti-protein antibody or a secondary antibody which binds theanti-protein antibody. In addition, other high affinity ligands may beused. Immunoassays which can be used include e.g. ELISAs, Western blots,and other techniques known to those of ordinary skill in the art (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999 and Edwards R,Immunodiagnostics: A Practical Approach, Oxford University Press,Oxford; England, 1999). All these detection techniques may also beemployed in the format of microarrays, protein-arrays, antibodymicroarrays, tissue microarrays, electronic biochip or protein-chipbased technologies (see Schena M., Microarray Biochip Technology, EatonPublishing, Natick, Mass., 2000).

Further, NALP7 nucleic acid-containing sequences may be amplified usingknown methods (e.g. polymerase chain reaction [PCR]) prior to or inconjunction with the detection methods noted herein. Examples of PCRprimers for amplification of NALP7 sequences are provided in theExamples herein. The design of various primers for such amplification isknown in the art.

The detection methods herein may also be performed in an assay utilizinga substrate having detection reagents attached thereto at discretelocations, such as a nucleic acid microarray. The invention furtherprovides a substrate comprising an isolated altered NALP7 nucleic aciddescribed herein attached thereto.

The invention further provides a nucleic acid, e.g., a probe, capable ofspecifically hybridizing to the altered NALP7 nucleotide sequence andnot to (or to a lesser extent to) a corresponding wild-type NALP7nucleic acid sequence (under comparable hybridization conditions). Suchhybridization may be under moderately stringent, or preferablystringent, conditions, e.g. as noted below. Such a probe or pluralitythereof may in embodiments be attached to a solid substrate, as notedabove.

The invention further provides (a) nucleic acid primer(s) (e.g. anamplification pair) specific for the alteration, wherein the primer(s)produce(s) an amplified product if the alteration is present and doesnot produce the same amplified product when a corresponding wild-typeNALP7 nucleic acid sequence is used as a template for amplification.

The invention further provides an isolated nucleic acid encoding theabove-mentioned altered NALP7 polypeptide. The invention furtherprovides an isolated altered NALP7 nucleic acid comprising the abovenoted alteration. The invention further provides an isolated,substantially pure, or recombinant polypeptide encoded by theabove-mentioned nucleic acid, as well as fusion proteins comprising thepolypeptide and an additional polypeptide sequence (e.g. a heterologouspolypeptide sequence). The invention further provides an isolated,substantially pure, or recombinant polypeptide comprising the abovenoted alteration. The invention further provides isolated nucleic acidshaving a nucleotide sequence which is substantially identical to theabove-noted altered NALP7 nucleic acid of the invention. The inventionfurther provides an isolated, substantially pure, or recombinantpolypeptide having an amino acid sequence which is substantiallyidentical to the above-noted altered NALP7 polypeptide of the invention.

“Altered NALP7 nucleic acid” or “altered NALP7 gene” as used hereinrefer to a nucleic acid comprising a nucleotide sequence which differsfrom a wild-type NALP7 nucleotide sequence in that it comprises analteration as noted herein. “NALP7 nucleic acid”, “NALP7 gene”,“wild-type NALP7 nucleic acid” or “wild-type NALP7 gene” as used hereinrefer to a nucleic acid comprising a nucleotide sequence encoding aNALP7 polypeptide or protein. “NALP7 polypeptide”, “NALP7 protein”,“wild-type NALP7 polypeptide” or “wild-type NALP7 protein” as usedherein refer to a polypeptide comprising the amino acid sequence of aNALP7 polypeptide present in subjects not suffering from a reproductivecondition, and having NALP7 activity. Examples of nucleotide sequencesof human wild-type NALP7 genes or nucleic acids are set forth in FIGS.6-8 and SEQ ID NOs: 1, 2 and 4. Examples of amino acid sequences ofhuman wild-type NALP7 polypeptides or proteins are set forth in FIGS. 7and 8 and SEQ ID NOs: 3 (980 amino acid isoform) and 5 (1009 amino acidisoform).

“Homology” and “homologous” refers to sequence similarity between twopeptides or two nucleic acid molecules. Homology can be determined bycomparing each position in the aligned sequences. A degree of homologybetween nucleic acid or between amino acid sequences is a function ofthe number of identical or matching nucleotides or amino acids atpositions shared by the sequences. As the term is used herein, a nucleicacid sequence is “homologous” to another sequence if the two sequencesare “substantially identical”, as used herein, and the functionalactivity of the sequences is conserved (as used herein, the term‘homologous’ does not infer evolutionary relatedness). Two nucleic acidsequences are considered “substantially identical” if, when optimallyaligned (with gaps permitted), they share at least about 50% sequencesimilarity or identity, or if the sequences share defined functionalmotifs. In alternative embodiments, sequence similarity in optimallyaligned substantially identical sequences may be at least 60%, 70%, 75%,80%, 85%, 90% or 95%. As used herein, a given percentage of homologybetween sequences denotes the degree of sequence identity in optimallyaligned sequences. The invention thus further provides a nucleic acidcomprising a nucleotide sequence having at least 60%, 70%, 75%, 80%,85%, 90% or 95% identity with any of SEQ ID Nos 6-42, or with an alteredversion of any of SEQ ID NOs 1, 2 and 4 (DNA) and SEQ ID NOs 3 and 5(polypeptide) comprising an alteration noted herein or any combinationof the alterations noted herein. An “unrelated” or “non-homologous”sequence shares less than 40% identity, though preferably less thanabout 25% identity, with any of SEQ ID NOs described herein.

Substantially complementary nucleic acids are nucleic acids in which thecomplement of one molecule is “substantially identical” to the othermolecule. Two nucleic acid or protein sequences are considered“substantially identical” if, when optimally aligned, they share atleast about 70% sequence identity. In alternative embodiments, sequenceidentity may for example be at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%. Optimal alignment of sequences forcomparisons of identity may be conducted using a variety of algorithms,such as the local homology algorithm of Smith and Waterman, 1981, Adv.Appl. Math 2: 482, the homology alignment algorithm of Needleman andWunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and thecomputerised implementations of these algorithms (such as GAP, BESTFIT,FASTA and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, Madison, Wis., U.S.A.). Sequence identity may also bedetermined using the BLAST algorithm, described in Altschul et al.,1990, J. Mol. Biol. 215:403-10 (using the published default settings).Software for performing BLAST analysis may be available through theNational Center for Biotechnology Information (through the internet athttp://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence that either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as theneighbourhood word score threshold. Initial neighbourhood word hits actas seeds for initiating searches to find longer HSPs. The word hits areextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction is halted when the following parameters are met: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram may use as defaults a word length (W) of 11, the BLOSUM62scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of bothstrands. One measure of the statistical similarity between two sequencesusing the BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. In alternativeembodiments of the invention, nucleotide or amino acid sequences areconsidered substantially identical if the smallest sum probability in acomparison of the test sequences is less than about 1, preferably lessthan about 0.1, more preferably less than about 0.01, and mostpreferably less than about 0.001.

An alternative indication that two nucleic acid sequences aresubstantially complementary is that the two sequences hybridize to eachother under moderately stringent, or preferably stringent, conditions.Examples of nucleic acid hybridization conditions are described furtherbelow.

The invention further provides a vector comprising the above-mentionednucleic acid and a replicon active in a host cell (e.g. replicativecloning vector). The invention further provides a vector comprising theabove-mentioned nucleic acid operably-linked to a transcriptionallyregulatory sequence (e.g. an expression vector).

The invention further provides a host cell transformed with theabove-mentioned vector.

The invention further provides an immunological reagent, such as anantibody, which exhibits different immunoreactivity with an alteredNALP7 polypeptide, i.e., comprising the above-noted alteration, relativeto a wild-type NALP7 polypeptide.

As noted above, an isolated nucleic acid, for example a nucleic acidsequence encoding a polypeptide of the invention, or homolog, fragmentor variant thereof, may further be incorporated into a vector, such as arecombinant expression vector. In an embodiment, the vector willcomprise transcriptional regulatory sequences or a promoteroperably-linked to a nucleic acid comprising a sequence capable ofencoding a peptide compound, polypeptide or domain of the invention. Afirst nucleic acid sequence is “operably-linked” with a second nucleicacid sequence when the first nucleic acid sequence is placed in afunctional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably-linked to a coding sequence if thepromoter affects the transcription or expression of the codingsequences. Generally, operably-linked DNA sequences are contiguous and,where necessary to join two protein coding regions, in reading frame.However, since for example enhancers generally function when separatedfrom the promoters by several kilobases and intronic sequences may be ofvariable lengths, some polynucleotide elements may be operably-linkedbut not contiguous. “Transcriptional regulatory sequence/element” is ageneric term that refers to DNA sequences, such as initiation andtermination signals, enhancers, and promoters, splicing signals,polyadenylation signals which induce or control transcription of proteincoding sequences with which they are operably-linked. “Promoter” refersto a DNA regulatory region capable of binding directly or indirectly toRNA polymerase in a cell and initiating transcription of a downstream(3′ direction) coding sequence. For purposes of the present invention,the promoter is bound at its 3′ terminus by the transcription initiationsite and extends upstream (5′ direction) to include the minimum numberof bases or elements necessary to initiate transcription at levelsdetectable above background. Within the promoter will be found atranscription initiation site (conveniently defined by mapping with S1nuclease), as well as protein binding domains (consensus sequences)responsible for the binding of RNA polymerase. Eukaryotic promoters willoften, but not always, contain “TATA” boses and “CCAT” boxes.Prokaryotic promoters contain Shine-Dalgarno sequences in addition tothe −10 and −35 consensus sequences.

As used herein, “nucleic acid molecule”, refers to a polymer ofnucleotides. Non-limiting examples thereof include DNA (i.e. genomicDNA, cDNA) and RNA molecules (i.e. mRNA). The nucleic acid molecule canbe obtained by cloning techniques or synthesized. DNA can bedouble-stranded or single-stranded (coding strand or non-coding strand[antisense]).

The term “recombinant DNA” as known in the art refers to a DNA moleculeresulting from the joining of DNA segments. This is often referred to asgenetic engineering.

The terminology “amplification pair” refers herein to a pair ofoligonucleotides (oligos) of the present invention, which are selectedto be used together in amplifying a selected nucleic acid sequence byone of a number of types of amplification processes, preferably apolymerase chain reaction. Other types of amplification processesinclude ligase chain reaction, strand displacement amplification, ornucleic acid sequence-based amplification. As commonly known in the art,the oligos are designed to bind to a complementary sequence underselected conditions. Accordingly, the invention further provides anamplification pair capable of amplifying an altered NALP7 nucleic acid,a wild-type NALP7 nucleic acid, or a fragment of an altered NALP7nucleic acid or a wild-type NALP7 nucleic acid. Examples of suitableamplification pairs are set forth in Example 6 below, whereby anysuitable combination of forward (fwd) and reverse (rev) primers for agiven region are shown (both those utilized for PCR and sequencing maybe used as an amplification pair). For example: For Exon 1,representative amplification pairs include SEQ ID NOs: 6 and 7, and SEQID NOs: 6 and 35. For Exon 2, representative amplification pairs includeSEQ ID NOs: 8 and 9, and SEQ ID NOs: 8 and 36. For Exon 3,representative amplification pairs include SEQ ID NOs: 10 and 11, andSEQ ID NOs: 10 and 37. For Exon 4, representative amplification pairsinclude SEQ ID NOs: 12 and 13, SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and17, and SEQ ID NOs: 18 and 19. For Exon 5, representative amplificationpairs include SEQ ID NOs: 20 and 21, and SEQ ID NOs: 20 and 38. For Exon6, a representative amplification pair is SEQ ID NOs: 22 and 23. ForExon 7, representative amplification pairs include SEQ ID NOs: 24 and25, and SEQ ID NOs: 39 and 25. For Exon 8, representative amplificationpairs include SEQ ID NOs: 26 and 27, and SEQ ID NOs: 41 and 42. For Exon9, a representative amplification pair is SEQ ID NOs: 28 and 29. ForExon 10, representative amplification pairs include SEQ ID NOs: 30 and31, and SEQ ID NOs: 30 and 40. For Exon 11, a representativeamplification pair is SEQ ID NOs: 32 and 33. For the region comprisingthe IVS3+1 G>A mutation described herein, a representative amplificationpair is SEQ ID NOs: 10 and 34.

Oligonucleotide probes or primers of the present invention may be of anysuitable length, depending on the particular assay format and theparticular needs and targeted sequences employed. In general, theoligonucleotide probes or primers are at least 12 nucleotides in length,preferably between 15 and 24 molecules, and they may be adapted to beespecially suited to a chosen nucleic acid amplification system. Ascommonly known in the art, the oligonucleotide probes and primers can bedesigned by taking into consideration the melting point of hybridizationthereof with its targeted sequence (see below and in Sambrook et al.,1989, Molecular Cloning—A Laboratory Manual, 2nd Edition, CSHLaboratories; Ausubel et al., 1989, in Current Protocols in MolecularBiology, John Wiley & Sons Inc., N.Y.).

“Nucleic acid hybridization” refers generally to the hybridization oftwo single-stranded nucleic acid molecules having complementary basesequences, which under appropriate conditions will form athermodynamically favored double-stranded structure. Examples ofhybridization conditions can be found in the two laboratory manualsreferred above (Sambrook et al., 1989, supra and Ausubel, et al. (eds),1989, Current Protocols in Molecular Biology, Vol. 1, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., New York) and arecommonly known in the art. Hybridization to filter-bound sequences undermoderately stringent conditions may, for example, be performed in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel, et al. (eds), 1989,Current Protocols in Molecular Biology, Vol. 1, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).Alternatively, hybridization to filter-bound sequences under stringentconditions may, for example, be performed in 0.5 M NaHPO₄, 7% SDS, 1 mMEDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel,et al. (eds), 1989, supra). In other examples of hybridization, anitrocellulose filter can be incubated overnight at 65° C. with alabeled probe in a solution containing 50% formamide, high salt (5×SSCor 5×SSPE), 5×Denhardt's solution, 1% SDS, and 100 μg/ml denaturedcarrier DNA (i.e. salmon sperm DNA). The non-specifically binding probecan then be washed off the filter by several washes in 0.2×SSC/0.1% SDSat a temperature which is selected in view of the desired stringency:room temperature (low stringency), 42° C. (moderate stringency) or 65°C. (high stringency). Hybridization conditions may be modified inaccordance with known methods depending on the sequence of interest (seeTijssen, 1993, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York). The selected temperature isbased on the melting temperature (Tm) of the DNA hybrid (Sambrook et al.1989, supra). Generally, stringent conditions are selected to be about5° C. lower than the thermal melting point for the specific sequence ata defined ionic strength and pH. Of course, RNA-DNA hybrids can also beformed and detected. In such cases, the conditions of hybridization andwashing can be adapted according to well-known methods by the person ofordinary skill. Stringent conditions will be preferably used (Sambrooket al., 1989, supra).

Probes or primers of the invention can be utilized with naturallyoccurring sugar-phosphate backbones as well as modified backbonesincluding phosphorothioates, dithionates, alkyl phosphonates andα-nucleotides and the like. Modified sugar-phosphate backbones aregenerally taught by Miller, 1988, Ann. Reports Med. Chem. 23:295 andMoran et al., 1987, Nucleic acid molecule. Acids Res., 14:5019. Probesor primers of the invention can be constructed of either ribonucleicacid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.

The types of detection methods in which probes can be used includeSouthern blots (DNA detection), dot or slot blots (DNA, RNA), andNorthern blots (RNA detection). Although less preferred, labeledproteins could also be used to detect a particular nucleic acid sequenceto which it binds.

Although the present invention is not specifically dependent on the useof a label for the detection of a particular nucleic acid sequence, sucha label might be beneficial, by increasing the sensitivity of thedetection. Furthermore, it enables automation (the same can also be saidof detection of proteins using ligands such as antibodies). Probes canbe labeled according to numerous well-known methods (Sambrook et al.,1989, supra). Non-limiting examples of detectable markers includeligands, fluorophores, chemiluminescent agents, enzymes, and antibodies.Other detectable markers for use with probes, which can enable anincrease in sensitivity of the method of the invention, include biotinand radionucleotides. It will be understood by the person of ordinaryskill that the choice of a particular label dictates the manner in whichit is bound to the probe.

As commonly known, radioactive nucleotides can be incorporated intoprobes of the invention by several methods. Non-limiting examplesthereof include kinasing the 5′ ends of the probes using gamma ³²P ATPand polynucleotide kinase, using the Klenow fragment of Pol I of E. coliin the presence of radioactive dNTP (e.g. uniformly labeled DNA probeusing random oligonucleotide primers in low-melt gels), using the SP6/T7system to transcribe a DNA segment in the presence of one or moreradioactive NTP, and the like.

As used herein, “oligonucleotides” or “oligos” define a molecule havingtwo or more nucleotides (ribo or deoxyribonucleotides). The size of theoligo will be dictated by the particular situation and ultimately on theparticular use thereof and adapted accordingly by the person of ordinaryskill. An oligonucleotide can be synthesized chemically or derived bycloning according to well-known methods.

As used herein, a “primer” defines an oligonucleotide which is capableof annealing to a target sequence, thereby creating a double strandedregion which can serve as an initiation point for DNA synthesis undersuitable conditions.

Amplification of a selected, or target, nucleic acid sequence may becarried out by a number of suitable methods. See generally Kwoh et al.,1990, Am. Biotechnol. Lab. 8:14-25. Numerous amplification techniqueshave been described and can be readily adapted to suit particular needsof a person of ordinary skill. Non-limiting examples of amplificationtechniques include polymerase chain reaction (PCR), ligase chainreaction (LCR), strand displacement amplification (SDA),transcription-based amplification, the Qβ replicase system and NASBA(Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi etal., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol.Biol., 28:253-260; and Sambrook et al., 1989, supra). Preferably,amplification will be carried out using PCR.

Polymerase chain reaction (PCR) is carried out in accordance with knowntechniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;and 4,965,188 (the disclosures of all three U.S. patent are incorporatedherein by reference). In general, PCR involves, a treatment of a nucleicacid sample (e.g., in the presence of a heat stable DNA polymerase)under hybridizing conditions, with one oligonucleotide primer for eachstrand of the specific sequence to be detected. An extension product ofeach primer which is synthesized is complementary to each of the twonucleic acid strands, with the primers sufficiently complementary toeach strand of the specific sequence to hybridize therewith. Theextension product synthesized from each primer can also serve as atemplate for further synthesis of extension products using the sameprimers. Following a sufficient number of rounds of synthesis ofextension products, the sample is analyzed to assess whether thesequence or sequences to be detected are present. Detection of theamplified sequence may be carried out by visualization following EtBrstaining of the DNA following gel electrophoresis, or using a detectablelabel in accordance with known techniques, and the like. For a review onPCR techniques (see PCR Protocols, A Guide to Methods andAmplifications, Michael et al. Eds, Acad. Press, 1990).

Ligase chain reaction (LCR) is carried out in accordance with knowntechniques (Weiss, 1991, Science 254:1292). Adaptation of the protocolto meet the desired needs can be carried out by a person of ordinaryskill. Strand displacement amplification (SDA) is also carried out inaccordance with known techniques or adaptations thereof to meet theparticular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA89:392-396; and ibid., 1992, Nucleic Acids Res. 20:1691-1696).

The term “vector” is commonly known in the art and defines a plasmidDNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicleinto which DNA of the present invention can be cloned. Numerous types ofvectors exist and are well known in the art.

The term “expression” defines the process by which a gene is transcribedinto mRNA (transcription), the mRNA is then being translated(translation) into one polypeptide (or protein) or more.

The recombinant expression vector of the present invention can beconstructed by standard techniques known to one of ordinary skill in theart and found, for example, in Sambrook et al. (supra). A variety ofstrategies are available for ligating fragments of DNA, the choice ofwhich depends on the nature of the termini of the DNA fragments and canbe readily determined by persons skilled in the art. The vectors of thepresent invention may also contain other sequence elements to facilitatevector propagation (e.g. a replicon) and selection in bacteria and hostcells. In addition, the vectors of the present invention may comprise asequence of nucleotides for one or more restriction endonuclease sites.Coding sequences such as for selectable markers and reporter genes arewell known to persons skilled in the art.

A recombinant expression vector comprising a nucleic acid sequence ofthe present invention may be introduced into a host cell, which mayinclude a living cell capable of expressing the protein coding regionfrom the defined recombinant expression vector. The living cell mayinclude both a cultured cell and a cell within a living organism.Accordingly, the invention also provides host cells containing therecombinant expression vectors of the invention. The terms “host cell”and “recombinant host cell” are used interchangeably herein. Such termsrefer not only to the particular subject cell but to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

Vector DNA can be introduced into cells via conventional transformationor transfection techniques. The terms “transformation” and“transfection” refer to techniques for introducing foreign nucleic acidinto a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection,electroporation, microinjection and viral-mediated transfection.Suitable methods for transforming or transfecting host cells can forexample be found in Sambrook et al. (supra), and other laboratorymanuals.

Recombinant production is useful for the preparation of large quantitiesof the protein encoded by the DNA sequence of interest. The protein canbe purified according to standard protocols that take advantage of theintrinsic properties thereof, such as size and charge (i.e. SDS gelelectrophoresis, gel filtration, centrifugation, ion exchangechromatography . . . ). In addition, the protein of interest can bepurified via affinity chromatography using polyclonal or monoclonalantibodies or other affinity-based systems (e.g. using a suitableincorporated “tag” in the form of a fusion protein and its correspondingligand). Suitable recombinant systems include prokaryotic and eukaryoticexpression systems, which are known in the art.

The term “allele” defines an alternative form of a gene which occupies agiven locus on a chromosome.

As commonly known, a “mutation” is a detectable change in the geneticmaterial which can be transmitted to a daughter cell. As well known, amutation can be, for example, a detectable change in one or moredeoxyribonucleotide. For example, nucleotides can be added, deleted,substituted for, inverted, or transposed to a new position. Spontaneousmutations and experimentally induced mutations exist. A mutantpolypeptide can be encoded from a mutant nucleic acid molecule. Inaddition, mutant proteins can be produced through aberrant events duringreplication, transcription and/or translation. Frameshifting (theswitching from a particular reading frame to another) is such amechanism that can modify the sequence of the translated protein.

A compound is “substantially pure” when it is separated from thecomponents that naturally accompany it. Typically, a compound issubstantially pure when it is at least 60%, more generally 75% or over90%, by weight, of the total material in a sample. Thus, for example, apolypeptide that is chemically synthesized or produced by recombinanttechnology will generally be substantially free from its naturallyassociated components. A nucleic acid molecule is substantially purewhen it is not immediately contiguous with (i.e., covalently linked to)the coding sequences with which it is normally contiguous in thenaturally occurring genome of the organism from which the DNA of theinvention is derived. A substantially pure compound can be obtained, forexample, by extraction from a natural source; by expression of arecombinant nucleic acid molecule encoding a polypeptide compound; or bychemical synthesis. Purity can be measured using any appropriate methodsuch as column chromatography, gel electrophoresis, HPLC, etc.

As used herein, the terms “molecule”, “compound”, “agent”, or “ligand”are used interchangeably and broadly to refer to natural, synthetic orsemi-synthetic molecules or compounds. The term “molecule” thereforedenotes for example chemicals, macromolecules, cell or tissue extracts(from plants or animals) and the like. Non-limiting examples ofmolecules include nucleic acid molecules, peptides, antibodies,carbohydrates and pharmaceutical agents. The agents can be selected andscreened by a variety of means including random screening, rationalselection and by rational design using for example protein or ligandmodelling methods such as computer modelling.

A further aspect of the invention provides an antibody that recognizesan altered NALP7 polypeptide of the invention. Antibodies may berecombinant, e.g., chimeric (e.g., constituted by a variable region ofmurine origin associated with a human constant region), humanized (ahuman immunoglobulin constant backbone together with hypervariableregion of animal, e.g., murine, origin), and/or single chain. Bothpolyclonal and monoclonal antibodies may also be in the form ofimmunoglobulin fragments, e.g., F(ab)′₂ Fab or Fab′ fragments. Theantibodies of the invention are of any isotype, e.g., IgG or IgA, andpolyclonal antibodies are of a single isotype or a mixture of isotypes.In general, techniques for preparing antibodies (including monoclonalantibodies and hybridomas) and for detecting antigens using antibodiesare well known in the art.

Antibodies against the altered NALP7 polypeptide of the presentinvention are generated by immunization of a mammal with a partiallypurified fraction comprising altered NALP7 polypeptide. Such antibodiesmay be polyclonal or monoclonal. Methods to produce polyclonal ormonoclonal antibodies are well known in the art. For a review, seeHarlow and Lane (1988) and Yelton et al. (1981), both of which areherein incorporated by reference. For monoclonal antibodies, see Kohlerand Milstein (1975), and Campbell, 1984, In “Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology”, Elsevier Science Publisher, Amsterdam, The Netherlands.

The antibodies of the invention, which are raised to a partiallypurified fraction comprising altered NALP7 polypeptide of the invention,are produced and identified using standard immunological assays, e.g.,Western blot analysis, dot blot assay, or ELISA (see, e.g., Coligan etal. (1994), herein incorporated by reference). The antibodies are usedin diagnostic methods to detect the presence of a altered NALP7polypeptide and activity in a sample, such as a tissue or body fluid.The antibodies are also used in affinity chromatography for obtaining apurified fraction comprising the altered NALP7 polypeptide and activityof the invention.

Accordingly, a further aspect of the invention provides (i) a reagentfor detecting the presence of altered NALP7 polypeptide and activity ina tissue or body fluid; and (ii) a diagnostic method for detecting thepresence of altered NALP7 polypeptide and activity in a tissue or bodyfluid, by contacting the tissue or body fluid with an antibody of theinvention, such that an immune complex is formed, and by detecting suchcomplex to indicate the presence of altered NALP7 polypeptide andactivity in the sample or the organism from which the sample is derived.

Those skilled in the art will readily understand that the immune complexis formed between a component of the sample and the antibody, and thatany unbound material is removed prior to detecting the complex. It isunderstood that an antibody of the invention is used for screening asample, such as, for example, blood, plasma, lymphocytes, cerebrospinalfluid, urine, saliva, epithelia and fibroblasts, for the presence of analtered NALP7 polypeptide.

For diagnostic applications, the reagent (i.e., the antibody of theinvention) is either in a free state or immobilized on a solid support,such as a tube, a bead, or any other conventional support used in thefield. Immobilization is achieved using direct or indirect means. Directmeans include passive adsorption (non-covalent binding) or covalentbinding between the support and the reagent. By “indirect means” ismeant that an anti-reagent compound that interacts with a reagent isfirst attached to the solid support. Indirect means may also employ aligand-receptor system, for example, where a molecule such as a vitaminis grafted onto the reagent and the corresponding receptor immobilizedon the solid phase. This is illustrated by the biotin-streptavidinsystem. Alternatively, a peptide tail is added chemically or by geneticengineering to the reagent and the grafted or fused product immobilizedby passive adsorption or covalent linkage of the peptide tail.

The present invention also relates to a kit for diagnosing a conditionof the female reproductive system, or a predisposition to contractingsame, comprising suitable means to detect the above-mentionedalteration, such as a probe, primer (or primer pair), or immunologicalreagent (e.g. antibody) in accordance with the present invention. Forexample, a compartmentalized kit in accordance with the presentinvention includes any kit in which reagents are contained in separatecontainers. Such containers include small glass containers, plasticcontainers or strips of plastic or paper. Such containers allow theefficient transfer of reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers may for example include a container which will accept thetest sample (DNA, protein or cells), a container which contains theprimers used in the assay, containers which contain enzymes, containerswhich contain wash reagents, and containers which contain the reagentsused to detect the indicator products. In an embodiment the kit furthercomprises instructions for diagnosing a condition of the femalereproductive system, or a predisposition to contracting same.

In another aspect, the invention relates to the use of a NALP7-defectiveimmune cell (e.g., having a mutated [e.g., comprising an alterationdescribed herein] or disrupted NALP7 gene, lacking a NALP7 gene, orhaving been treated or engineered for decreased NALP7 expression orfunction [e.g., via NALP7-targeted RNA interference or antisenseoligonucleotides]) in screening assays that may be used to identifycompounds that are capable of restoring defective immune functionassociated with a female reproductive condition noted herein. In someembodiments, such an assay may comprise the steps of (a) providing atest compound; (b) providing a a NALP7-defective immune cell; and (c)determining cytokine release in the presence versus the absence of thetest compound. An increase in cytokine release in the presence versusthe absence of the compound is indicative that the compound is capableof restoring defective immune function associated with a femalereproductive condition.

The above-mentioned immune cell may be for example a peripheral bloodmononuclear cell (PBMC), lymphocyte or monocyte. The above-mentionedcytokine may be for example interleukin-1β (IL-1β) or TNF alpha (TNFα).

Cytokine release may in embodiments be measured in response to asuitable stimulus, such as in response to bacterial lipopolysaccharide(LPS) as described in the Examples below.

The above-noted assays may be applied to a single test compound or to aplurality or “library” of such compounds (e.g. a combinatorial library).Any such compounds may be utilized as lead compounds and furthermodified to improve their therapeutic, prophylactic and/orpharmacological properties.

Such assay systems may comprise a variety of means to enable andoptimize useful assay conditions. Such means may include but are notlimited to: suitable buffer solutions, for example, for the control ofpH and ionic strength and to provide any necessary components foroptimal stability (e.g. protease inhibitors) of assay components,temperature control means for optimal activity and or stability of assaycomponents, and detection means to enable the detection of the indicatorproduct. A variety of such detection means may be used, including butnot limited to one or a combination of the following: radiolabelling(e.g. ³²P, ¹⁴C, ³H), antibody-based detection, fluorescence,chemiluminescence, spectroscopic methods (e.g. generation of a productwith altered spectroscopic properties), various reporter enzymes orproteins (e.g. horseradish peroxidase, green fluorescent protein),specific binding reagents (e.g. biotin/(streptavidin)), and others.

The present invention is illustrated in further details by the followingnon-limiting examples.

EXAMPLES Example 1 Methods

Mutation screening and analysis. Genomic structure of the screened geneswere obtained from publicly available databases(http://genome.ucsc.edu/) and the primers flanking predicted exons,exon/intron boundaries and 5′ and 3′UTRs were designed using PrimerSelect v5.05 (DNAStar). Exons were PCR amplified, visualized on 2%agarose gels stained with ethidium bromide, and sequenced directly usinga 3730XL DNA Analysis System (Applied Biosystems). Sequences werealigned using SeqManII v5.05 and screened for mutations.

RT-PCR. Total RNAs was extracted from EBV transformed lymphoblast celllines using Trizol (Invitrogen). Three micrograms of total RNA werereverse-transcribed using 200 units of M-MLV Reverse Transcriptase(Invitrogen) with RNA Guard RNase Inhibitor (Amersham) in a total volumeof 50 μl. Five microliters of this preparation were then PCR amplifiedaccording to standard protocols. Sequencing of cDNA fragments were doneon direct PCR products or after purification of the appropriate bandsand cloning using the TOPO TA™ Cloning Kit (Invitrogen).

Example 2 Fine Mapping of the HM Candidate Region

To identify the defective gene associated with recurrent HMs, applicantscreened all the predicted exons of the 53 genes present in the reportedhypothetical 1.1-Mb minimal interval (Sensi et al.; Hodges et al.).However, applicant did not find any mutations in this region. Applicantthus confirmed that the proximal boundary of the reported 1.1-Mb minimalinterval is incorrect. Using a proximal boundary identified in familyMoLb1 as a 1.29-Mb region between D19S924 and D19S926 (Moglabey et al.,1999), applicant identified herein nine new polymorphic markers from theavailable genomic DNA sequences and genotyped them in MoLb1. Thisanalysis defined marker 11515-31 as the proximal boundary of the HMcandidate region (FIG. 1). This new definition of the proximal boundaryadded a cluster of killer-cell immunoglobulin-like receptors (KIR) genes(7 to 14 genes depending on haplotypes), two KIR-related genes, NCR1,and FCAR, and NALP7. Genotyping of an additional family, MoPa61previously reported by Mazhar and Janjua (1995) with 23 polymorphicmarkers from 19q13.4 demonstrated its linkage to this region and defineda single nucleotide polymorphism (SNP) located 16 bases upstream exon 3of gene COX6B2 (NM_(—)144613) as the distal boundary of the minimal HMcandidate region (FIG. 2). Based on data from MoLb1 and MoPa61,applicant fine mapped the HM candidate region to 0.65-Mb between11515-31 and COX6B2ex3.

Example 3 Mutation Analyses

By screening the additional genes identified by the new definition ofthe proximal boundary herein, applicant identified in NALP7 (also calledPYPAF3) two different mutations affecting the invariant G of the GTsplice donor site at the junction of exon 3/intron 3 (IVS3+1G>A) in apatient from MoLb1 (FIG. 3 a) and at the junction of exon 7/intron 7(IVS7+1G>A) in a patient from MoPa61 (FIG. 4). The mutation in familyMoLb1 abolishes a recognition site for the restriction enzyme BstN1thatapplicant used to detect the mutation in the other members of the family(FIG. 3 b) and in 100 control women (with 5 to 16 children) from variousethnic groups. In family MoPa61, IVS7+1G>A, the mutation wasinvestigated in the other members of the family and controls by DNAsequencing (FIG. 4). Both mutations segregate with the disease phenotypein their respective families and were not found in the 200 controlchromosomes screened. In family MoGe2, applicant identified in exon 5, aC to T change substituting an arginine for a tryptophan at amino acid693, R693W (FIG. 4), a conserved residue in chimpanzee and cow NALP7 aswell as in human, cow, and dog NALP2. By DNA sequencing, it was foundthat this change co-segregates with the disease status in MoGe2 and isnot present on 274 chromosomes from control women with five to sixteenchildren.

To assess the role of NALP7 in recurrent molar pregnancies occurring insingle-family members that are not homozygous at 19q13.4 markers andcould not be investigated for linkage to 19q13.4 (because of the absenceof other female siblings with known pregnancy outcomes in the family),applicant screened NALP7 in eight such cases and identified additionalset of five new mutations, C399Y, E99X, C84Y, K379N, and D657V that werenot found in controls. Mutations, clinical data, and coding DNApolymorphisms found in the different families and patients aresummarized in Table 1.

TABLE 1 Summary of mutations, ethnic origin, and clinical manifestationsof the patients Nucleotide Amino acid Clinical manifestations FamilyPopulation Location change change and outcomes Reference Familial casesof recurrent moles MoLb1 Lebanese Intron 3 IVS3 + 1G > A NP, SB, SA,CHM, PHM, Seoud et al, 1995, Helwani et al., PTD, preeclampsia 1999MoPa61 Pakistani Intron 7 IVS7 + 1G > A SA, CHM Mazhar and Janjua 1995MoGe2 German Exon 5 2077C > T R693W CHM Kircheisen and Ried, 1994 MoCh76Chinese Exon 3 365G > T E99X SB, CHM Present study Exon 5 2040A > TD657V Single family member with recurrent moles MoCh71 Chinese Exon 2321G > A C84Y 2 CHMs Present study Heterozygous MoCh73 Chinese Exon 41207G > C K379N 2 CHMs, 1 PHM Present study Heterozygous MoCa57 MoroccanExon 4 1266G > A C399Y SA, BO, TP + CHM Present study Heterozygous Withreference to Table 1, the phenotype of the conceptuses were as reportedin the original papers listed under Reference. Nucleotide positions aregiven according to RefSeq mRNA NM_206828, amino acid positions accordingto Q8WX94. NP, normal pregnancy; SB, stillbirth; SA, spontaneousabortion; CHM, complete hydatidiform mole, PHM, partial hydatidiformmole PTD, persistent trophoblastic disease; BO, blighted ovum; TP, twinpregnancy.

Example 4 Expression of NALP7 in Normal Tissues

A recent study has reported NALP7 expression in a broad range of normaladult tissues (Kinoshita et al., 2005). To investigate the role of NALP7in the pathology of moles, a disease caused by a maternal defectivegene, applicant investigated its transcription by RT-PCR in normal humanuterus and ovary using two combinations of primers located in exons 6and 8, and in exons 8 and 11. Applicant identified two NALP7transcripts, V1 and V2, in both tissues and also in EBV lymphoblastoidcell lines from normal subjects, and first trimester chorionic villi. ByDNA sequencing, we found that V1 and V2 are due to the exclusion orinclusion of exon 10, respectively.

Example 5 Effect of the Splice Mutations on NALP7 Transcription

The two splice mutations identified herein affect exons 3 and 7 (theseexons are present in all reported transcriptional isoforms). UsingGENSCAN (http://genes.mit.edu/GENSCAN.html), the splice mutationIVS3+1G>A was predicted to result in the skipping of exon 3, while SSPNN(http://www.fruitfly.org/seq_tools/splice.html) analysis, predicts theactivation and usage of a cryptic intronic splice site located 4-bpdownstream of exon 3. Using both programs, GENSCAN and SSPNN, the splicemutation IVS7+1G>A is predicted to lead to the skipping of exon 7.Primers located in exons 6 and 8 amplified a large fragment (˜1 kb) inthe three patients from MoPa61, that does not correspond to the size ofthe genomic fragment (2635 bp) between the two primers (FIG. 5). Thisfragment was observed only after reverse transcription and was notpresent in 5 normal control subjects. Applicant cloned and sequencedthis fragment and found it to correspond to the inclusion of the entireintron 7. The inclusion of intron 7 is expected to add next to exon 7only one amino acid, a serine, followed by a stop codon, TAA, leading toa shorter protein of 824 amino acids.

Example 6 Primers for PCR Amplification of Regions of NALP7

Exon 1: PCR Fwd: NALP7ex1a (SEQ ID NO: 6) GCCCAATTACAGCCAAATCCCTGAG Rev:NALP7ex1b Product Size: 604 bp (SEQ ID NO: 7) GGCCGAGGCAGACAGATTACCTAAASequencing NALP7ex1a (see SEQ ID NO: 6 above) NALP7Rev2 (SEQ ID NO: 35)TCCTTCCAGCATCCTCGCAC Exon 2: PCR NALP7ex2-fwd (SEQ ID NO: 8)ACCGTGCTGGGCCAGATTTTCAGT NALPex3-rev Product size: 777 bp (SEQ ID NOs:9; 11) GCAGAGGTTGCAATGAGCAGAGACG Sequencing NALP7ex2-fwd (see SEQ ID NO:8 above) NALP7ex2rev2 (SEQ ID NO: 36) ATGACCAGGACACCCCAGGTTCTA Exon3:PCR NALPex3-fwd (SEQ ID NO: 10) CCACCATGCCTGGCTGACACTTTAT NALPex3-revProduct size: 340 bp (SEQ ID NOs: 11; 9) GCAGAGGTTGCAATGAGCAGAGACGSequencing NALP7ex3-fwd (see SEQ ID NO: 10 above) NALP3ex2rev2 (SEQ IDNO: 37) CACCTTGCATGCTCTCAAACACCA Exon 4: 1-PCR NALP7ex4-1 fwd (SEQ IDNO: 12) GTAGTGGCTCCGTCTCTGCTCATTG NALP7ex4-1 rev Product Size: 737 bp(SEQ ID NO: 13) AGGCCATCGACCACGAACAGGATTC Sequencing NALP7ex4-1 fwd (seeSEQ ID NO: 12 above) NALP7ex4-1 rev (see SEQ ID NO: 13 above) 2-PCRNALPex4-2 fwd (SEQ ID NO: 14) GACGACGTCACTCTGAGAAACCAAC NALPex4-2 revProduct size: 757 bp (SEQ ID NO: 15) TGCAGAGGAAACGCAGGAACAGC SequencingNALPex4-2 fwd (see SEQ ID NO: 14 above) NALPex4-2 rev (see SEQ ID NO: 15above) 3-PCR NALP7ex4-3 fwd (SEQ ID NO: 16) TTTGCTGAAGAGGAAGATGTTACCCNALP7ex4-3 rev Product size: 722 bp (SEQ ID NO: 17)CGAGGCCGAATAAGAAGTGTCCTAC Sequencing NALPe7x4-3 fwd (see SEQ ID NO: 16above) NALP7ex4-3 rev (see SEQ ID NO: 17 above) 4-PCR NALP7ex4-4 fwd(SEQ ID NO: 18) GTGGGCGCAGATGTCCGTGTTC NALP7ex4-4 rev Product size: 803bp (SEQ ID NO: 19) CCTAATTGCCAAGTCGTGTCTCC Sequencing NALP7ex4-4 fwd(see SEQ ID NO: 18 above) NALP7ex4-4 rev (see SEQ ID NO: 19 above) Exon5: PCR NALP7ex-5 fwd (SEQ ID NO: 20) GGTCTCAGTTTCTAGCCCAAGTT NALP7ex-5rev (SEQ ID NO: 21) ACACGGTGAAAACCTGTCTGTGC Sequencing NALP7ex-5 fwd(see SEQ ID NO: 20 above) NALP7ex5rev2_Seq Product size: 839 bp (SEQ IDNO: 38) CAAGAAGCTTAGTCATCGTT Exon 6: PCR NALP7ex6-fwd (SEQ ID NO: 22)CCACTGCACCCGGCCAAGAACTT NALP7ex6-rev Product size: 597 bp (SEQ ID NO:23) GCTGGGGGCCACTGCTCTCAATC Sequencing NALP7ex6-fwd (see SEQ ID NO: 22above) NALP7ex6-rev (see SEQ ID NO: 23 above) Exon 7: PCR NALP7ex7-fwd(SEQ ID NO: 24) GATCACGCCTTTGCATTCCAGACTG NALP7ex7-rev Product size: 471bp (SEQ ID NO: 25) AACTCAGATGATCCGCCCACCTCTC Sequencing NALP7ex7Seq (SEQID NO: 39) AGCTGATAGGGTATACTCTG NALP7ex7-rev (see SEQ ID NO: 25 above)Exon 8: PCR NALP7ex8 fwd (SEQ ID NO: 26) AAAACAACACCTGTGTCCTGTGATGNALP7ex8 rev Product size: 849 bp (SEQ ID NO: 27)TTAACATGTTTCTACCTGTATCTGC NALP7ex8f2 (SEQ ID NO: 41)TGGCCATGATGACTCCCACAGG NALP7ex8r2 Product size: 418 bp (SEQ ID NO: 42)CCAGGTTTTTAAAAGTTACATTTG Sequencing NALP7ex8f2 (see SEQ ID NO: 26 above)NALP7ex8r2 (see SEQ ID NO: 27 above) Exon 9: PCR NALP7ex9-a (SEQ ID NO:28) CTTCACAGGGCGTTAGCCAGAGG NALP7ex9b Product size: 456 bp (SEQ ID NO:29) CCAGCCCGGGAAAGATGACAAGA Sequencing NALP7ex9-a (see SEQ ID NO: 28above) NALP7ex9b (see SEQ ID NO: 29 above) Exon 10: PCR NALP7ex10afwd(SEQ ID NO: 30) AAGGTGCTGGGGCTACAGGTGTCT NALP7ex10arev Product size: 787bp (SEQ ID NO: 31) GCCAACATGGTGAAACCCCTCTC Sequencing NALP7ex10afwd (seeSEQ ID NO: 30 above) NALP7ex10aseq_r (SEQ ID NO: 40) AAACCCATACCTGAGTATExon 11: PCR NALP7ex11 fwd (SEQ ID NO: 32) CTGTCCCCCAGAAAATCCCAAAAACNALP7ex11 rev Product size: 588 bp (SEQ ID NO: 33)CAACCGAATCATCCCTGAACTTC Sequencing NALP7ex11 fwd (see SEQ ID NO: 32above) NALP7ex11 rev (see SEQ ID NO: 33 above)To assess the IVS3+1G>A mutation using the restriction enzyme BstN1, thefollowing primers were used to amplify a 204 bp fragment that wasdigested with the enzyme:

(SEQ ID NO: 10) NALPex3-fwd CCACCATGCCTGGCTGACACTTTAT (SEQ ID NO: 34)NALPex3b2 CAAACACCAAACTCATGACCATA Product size: 204 bp

Example 8 Cytokine Release in Peripheral Mononuclear Cells from Patientswith Mutations in NALP7

The ability of peripheral blood mononuclear cells (PBMCs) harbouringhomozygous NALP7 mutations to secrete interleukin-1β (IL-1β) and TNFalpha (TNFα) in response to stimulation with bacteriallipopolysaccharide (LPS) was assessed. PBMCs were isolated from patientswith NALP7 mutations (MoLb1 with IVS3+1G>A and MoGe2 with R693W) andcontrol subjects using Ficoll gradient, stimulated with 100 ng/mL of LPSfor twenty hours and the supernatants were collected for cytokinequantification using ELISA. Applicant found that the concentration ofIL-1β and TNFα in the supernatant of patient' PBMCs was significantlylower than that of controls (FIGS. 9 and 10).

Although the present invention has been described hereinabove by way ofspecific embodiments thereof, it can be modified, without departing fromthe spirit and nature of the subject invention as defined in theappended claims. Throughout this application, various references arereferred to describe more fully the state of the art to which thisinvention pertains. The disclosures of these references are herebyincorporated by reference into the present disclosure.

REFERENCES

Helwani, M. N. et al., Hum Genet 105, 112-5 (1999).

Hodges, M. D., Rees, H. C., Seckl, M. J., Newlands, E. S. & Fisher, R.A., J Med Genet 40, e95 (2003).

Kinoshita, T., Wang, Y., Hasegawa, M., Imamura, R. & Suda, T., J BiolChem 280, 21720-5 (2005).

Kircheisen R, Ried T, Hum Reprod 9:1783 (1994).

Mazhar, S. & Janjua, S., J Pakistan Inst Med Sci 6, 383-6 (1995).

Moglabey, Y. B. et al., Hum Mol Genet 8, 667-71 (1999).

Okada, K. et al., Cancer Sci 95, 949-54 (2004).

Sensi, A. et al., Eur J Hum Genet 8, 641-4 (2000).

Seoud M, Khalil A, Frangieh A, Zahed L, Azar G, Nuwayri-Salti N., ObstetGynecol 86:692, (1995).

Silver, R. M., Lohner, W. S., Daynes, R. A., Mitchell, M. D. & Branch,D. W., Biol Reprod 50, 1108-12 (1994).

1. A method for diagnosing a predisposition for molar pregnancy in ahuman female subject, the method comprising detecting an alteration inthe sequence of a NALP7 gene or the sequence of its mRNA or encodedpolypeptide in a tissue sample from said subject relative to thesequence of a wild-type NALP7 gene or the sequence of its mRNA orencoded polypeptide, wherein said alteration is: a) a substitution of Gwith A at the splice donor site at the boundary of exon 3 and intron 3(IVS3+1G>A); b) a substitution of G with A at the splice donor site atthe boundary of exon 7 and intron 7 (IVS7+1G>A); c) a substitution of Cwith T corresponding to the first position of the codon for Arg 693 ofthe NALP7 polypeptide, resulting in a Arg to Trp substitution; d) asubstitution of G with A corresponding to the second position of thecodon for Cys 84 of the NALP7 polypeptide, resulting in a Cys to Tyrsubstitution; e) a substitution of G with A corresponding to the secondposition of the codon for Cys 399 of the NALP7 polypeptide, resulting ina Cys to Tyr substitution; f) a substitution of G with C correspondingto the third position of the codon for Lys 379 of the NALP7 polypeptide,resulting in a Lys to Asn substitution; g) a substitution of G with Tcorresponding to the first position of the codon for Glu 99 of the NALP7polypeptide, resulting in a substitution for a stop codon; and/or h) asubstitution of A with T corresponding to the second position of thecodon for Asp 657 of the NALP7 polypeptide, resulting in a Asp to Valsubstitution wherein if the NALP7 polypeptide is used for detecting saidalteration, said alteration is detected by sequencing of the NALP7polypeptide, and wherein said alteration indicates that the subject hasa predisposition for molar pregnancy.
 2. The method of claim 1, whereinsaid substitution of G with A at the splice donor site at the boundaryof exon 3 and intron 3 (IVS3+1G>A) is associated with a loss of acleavage site for the restriction endonuclease BstN1in the NALP7 gene.3. The method of claim 1, further comprising amplification of a nucleicacid sequence suspected of comprising the alteration in the sample priorto the detection of the alteration.
 4. The method of claim 1, whereindetection of the alteration in the sequence of the NALP7 gene or thesequence of its mRNA is performed using a method selected from: a)sequencing of the NALP7 nucleic acid sequence; b) hybridization of anucleic acid probe capable of specifically hybridizing to a NALP7nucleic acid sequence comprising the alteration and not to acorresponding wild-type NALP7 nucleic acid sequence; c) restrictionfragment length polymorphism analysis (RFLP); d) amplified fragmentlength polymorphism PCR (AFLP-PCR); and/or e) amplification of a nucleicacid fragment comprising a NALP7 nucleic acid sequence using a primerspecific for the alteration, wherein the primer produces an amplifiedproduct if the alteration is present and does not produce the sameamplified product when a corresponding wild-type NALP7 nucleic acidsequence is used as a template for amplification.
 5. The method of claim4, wherein said primer comprises a nucleotide sequence selected from SEQID NOs: 6-42.
 6. The method of claim 1, further comprising determiningcytokine release of an immune cell of said subject, wherein a decreasein cytokine release relative to a control level of cytokine release isfurther indicative that the subject suffers from or has a predispositionfor the reproductive condition.
 7. The method of claim 6, wherein thecontrol level is selected from an established standard and a level ofcytokine release of an immune cell comprising a wild-type NALP7 nucleicacid.
 8. The method of claim 6, wherein the immune cell is a lymphocyteor monocyte.
 9. The method of claim 6, wherein the immune cell is aperipheral blood mononuclear cell (PBMC).
 10. The method of claim 6,wherein the cytokine is selected from interleukin-1β (IL-1 β) and TNFalpha (TNFα).